Stereoselective Synthesis, Pro-resolution, and Anti-inflammatory Actions of RvD5n-3 DPA

The methyl ester of resolvin D5n-3 DPA, a lipid mediator biosynthesized from the omega-3 fatty acid n-3 docosapentaenoic acid, was stereoselectively prepared in 8% yield over 12 steps (longest linear sequence). The key steps for the introduction of the two stereogenic secondary alcohols were an organocatalyzed oxyamination and the Midland Alpine borane reduction. For the assembly of the carbon chain, the Sonogashira cross-coupling reaction and the Takai olefination were utilized. The physical properties, including retention time in liquid chromatography and tandem mass spectra, of the synthetic material were matched against material from human peripheral blood and mouse infectious exudates. Synthetic RvD5n-3 DPA, obtained just prior to biological experiments, displayed potent leukocyte-directed activities, upregulating the ability of neutrophils and macrophages to phagocytose bacteria, known as hallmark bioactions of specialized pro-resolving endogenous mediators.

T he acute inflammatory response is essential for protection against injury or pathogenic infections. 1 This self-limited and localized process is divided into an initiation and a resolution phase. 2 The resolution phase is essential to curtail inflammation and restore tissue homeostasis by elimination of harmful agents and cell debris. 3Normally, the acute inflammatory response is self-limited and resolves without interruption.However, if unresolved, a chronic inflammatory situation may result and develop further into human diseases, such as rheumatoid arthritis, ulcerative colitis, cardiovascular disorders, and Parkinson's and Alzheimer's diseases. 4It is now evident that actively induced biochemical pathways counterregulate inflammation and promote resolution at the site of infection or injury. 5Hence, resolution is an active rather than a passive process, as once believed, where oxygenated polyunsaturated lipid mediators are essential. 6These mediators, named specialized pro-resolving mediators (SPMs), have been isolated from diverse natural sources.−9 The ability of SPMs to govern inflammatory resolution processes is considered a biomedical paradigm shift. 10SPMs are nontoxic and potent agonists that stereoselectively activate G-protein coupled receptors (GPCRs). 11,12Examples of SPMs are the DHA-derived resolvins, protectins and maresins. 134−16 Some examples are shown in Figure 1.
Because SPMs enable resolution of inflammation without immunosuppression or toxic effects, these naturally occurring compounds have gained great interest in drug discovery. 10,11owever, such efforts depend on the stereoselective total synthesis of SPMs for further biological studies.Moreover, for configurational assignments, matching experiments using LC-MS/MS are in demand, as SPMs are formed in nano-to picogram amounts, too low for NMR configurational studies. 5,16,17s of today, stereoselective total syntheses of RvD1 n-3 DPA 18 and RvD2 n-3 DPA 19 have been achieved (Figure 1).Recently, it was reported that RvD5 n-3 DPA (1) binds to and activates GPR101, exerting tissue-protective actions during inflammatory arthritis. 15This SPM is formed after two consecutive lipoxygenation reactions, followed by hydroperoxide reduction by a peroxidase (Scheme 1).In the detailed anticipated biogenetic formation of 1, the first biosynthetic step in the presence of 15-lipoxygenase (LOX) forms 17(S)-HpDPA, while the second lipoxygenation step is catalyzed by 5-LOX.This product, 7(S),17(S)-diHpDPA, is then directly reduced to give SPM 1.Alternatively, an epoxide intermediate could be formed that is enzymatically hydrolyzed to either RvD1 n-3 DPA or RvD2 n-3 DPA (Scheme 1).
The DHA-derived SPM RvD5 (Figure 1) was isolated from inflammatory exudates, and its structure was established by UV data, LC-MS/MS experiments, 8 and total synthesis. 20,21RvD5 was first detected in leukocytes, brain cells, and glia cells, 8 and later it was also detected in several patient models. 22RvD5 given to mice infected with Escherichia coli has shown a significantly enhanced phagocyte containment of E. coli compared to mice infected with E. coli alone (160% increase). 23In addition, it showed protection from hypothermia and overall increased survival in E. coli-infected mice.RvD5 also reduced proinflammatory cytokines such as KC and TNF-α. 23Further studies revealed that RvD5 plays a role in downregulating a panel of inflammation-related genes, including NF-κB, phosphodiesterase 4B (PDE4B), and COX-2, which might contribute to their actions in enhancing phagocytosis and bacterial clearance in vivo. 23The biologically interesting effects reported for the DHA-derived SPMs spurred an interest in investigating the biosynthetic formation of n-3 DPA-derived SPMs. 7The congener RvD5, RvD5 n-3 DPA (1), was first reported in 2013.LC-MS/MS data, physical properties (MS and UV−vis data), and biosynthetic considerations enabled the tentative structure elucidation of 1 as depicted in Scheme 1. 7 This SPM contains two highly sensitive E,Z-dienes, both adjacent to stereogenic allylic alcohols that are prone to elimination reactions (Scheme 2).Based on our successful synthesis of RvE4, 24 the retrosynthetic proposal outlined in Scheme 2 seemed attractive to attempt.This analysis identified three key fragments, 3, 4, and 5, as suitable building blocks, wherein the linchpin 4 is commercially available.The chemically labile Z,Zskipped diene present in 1 and 2 should be introduced late in any total synthesis efforts.

■ RESULTS AND DISCUSSION
Despite the apparent similar structural features between RvE4 and RvD5 n-3 DPA (1), we experienced that SPM 1 is chemically more sensitive than RvE4.The same observations were also applied for some of the intermediates.However, the total synthesis was initiated with the preparation of the ω-3 fragment 3 from affordable cis-4-heptenal (6) (Scheme 3), utilizing a protocol developed by the MacMillan group 25 and essentially as earlier reported in our total synthesis of RvE4. 24This enantioselective, organocatalytic α-oxyamination reaction afforded diol 7 in 82% yield and 98% enantiomeric excess (ee).This diol was converted, via a highly E-selective Takai olefination in the last step, to vinyl iodide 3 (Scheme 3).Diastereomerically pure 3 was obtained after chromatographic purification.
The synthesis of vinyl iodide 5 was recently presented 19 and was resynthesized in order to investigate the outlined Sonogashira cross-coupling reactions. 26This reaction has also been successfully applied in total synthesis of SPMs, 27 including RvD5. 20he first reaction was performed with the linchpin 4 and catalytic amounts of Pd(PPh 3 ) 2 Cl 2 (5 mol %) and CuI (12 mol %), which provided a high yield of the coupled product 8 (Scheme 4).Due to the inherent lability of the diyne system in 8, TMS deprotection was accomplished utilizing the mild reaction conditions of AgNO 3 and KCN, 28 yielding the terminal alkyne 9 in 85% yield.The same Sonogashira cross-coupling conditions were then tried for the final carbon−carbon bond-forming reaction between alkyne 9 and vinyl iodide 3. Unfortunately, these reaction conditions afforded product 10 in a disappointingly low yield of 34%.Therefore, the classic Sonogashira coupling conditions, with Pd(PPh 3 ) 4 (3 mol %) and CuI (6 mol %), were attempted, but the coupled product 10 was not observed.However, vinyl iodide 3 was reisolated in 90% yield, while terminal alkyne 9 no longer could be detected in the reaction mixture.This indicated decomposition of the alkyne or undesired side reactions such as homocoupling of the alkyne and further decomposition.Nevertheless, no homocoupled byproduct was isolated, strengthening the notion of rapid decomposition of diyne 9.Such challenges were not observed in the preparation of RvE4. 24With this information, the first Sonogashira reaction conditions containing Pd(PPh 3 ) 2 Cl 2 (5 mol %) and CuI (11 mol %) was tried once more, with the equivalent of diyne 9 increased from 1.2 to 2.5.These conditions gave the desired coupled product 10 in an isolated 62% yield.
Then our tried-and-tested Z-selective Lindlar hydrogenation protocol was first attempted for the diyne system in 10, 25,29 but HRMS analyses of the reaction mixture revealed no reduction of the triple bonds.This was a surprise, as Rodriguez and Spur successfully used a Lindlar reduction in their total synthesis of RvD5, 20 although on a slightly different diyne than 10.A search in the literature gave interest in the Pd/BaSO 4 /quinoline hydrogenation protocol. 30,31These conditions were applied and showed rapid and selective reduction of the two triple bonds.Slight over-reduction was observed; however, isolation of product 11 in 71% yield was achieved after column chromatography.Removal of the two TBS groups was successfully accomplished by subjecting 11 to a catalytic amount of acetyl chloride in MeOH, 32 yielding the RvD5 n-3 DPA methyl ester 2 in 68% yield.These mild conditions were used instead of the more well-known tetra-n-butylammonium fluoride (TBAF) in THF due to reported byproduct formation and difficulties experienced during the purification process of RvE4. 24The ester 2 was obtained in 68% yield (97%, HPLC analysis) after chromatographic purification with NMR ( 1 H, 13 C, and COSY), HRMS, and UV data in accordance with the structure of 2 (Supporting Information).Analyses of the 1 H NMR data (J = 15.7 Hz for H8/H16, J = 15.0Hz for H9/H15, and J = 10.8Hz for H10/H11) and the COSY spectrum confirmed the olefin configurations of the two E,Z-dienes in 2 (Figure 1), which were further supported by the UV data (λ max = 242 nm; log ε = 4.64). 20e next evaluated whether the physical properties of the synthetic material matched those of biological RvD5 n-3 DPA .Using the free acid of the synthetic material 2, obtained just prior to analyses as described in the Experimental Section, due to the chemical sensitivity of SPMs, 5,33 we first evaluated whether the MS/MS spectrum matched that of the reference material.Here we observed that the MS/MS spectrum of synthetic 1 matched that of the reference material including key fragments such as m/ z 143, 199, and 263 (Figure 2).The UV data for synthetic 1 (λ max = 245 nm; log ε = 4.64, EtOH) were in accordance with the literature for biogenetic 1 7a,15 and its congener RvD5 (λ max = 245 nm). 8,20Similar data have been reported for RvE4, which contains the same olefin configuration. 24Moreover, UV data for E,E-configured oxygenated lipids show hypochromic effects. 34ecause we recently reported that RvD5 n-3 DPA is produced in human peripheral blood, 15 we next sought to compare the chromatographic properties of the synthetic material of 1 with those of endogenous RvD5 n-3 DPA from human serum.Using synthetic 1 we observed that in RP-LC-MS/MS experiments both the biological and synthetic RvD5 n-3 DPA (1) gave a peak in the multiple reaction monitoring (MRM) transition 361 > 199 with a retention time (t r ) of 13.5 min (Figure 3A).Furthermore, spiking of the biological material with 1 gave a single peak that eluted with a t r of 13.5 min (Figure 3A).Similar results were obtained with mouse infectious exudates where we observed a Journal of Natural Products peak in the 361 > 199 MRM transition with a t r of 13.5 min.Furthermore, spiking of the synthetic material of 1 into the biological material yielded a single peak in the 361 > 199 MRM transition with a t r of 13.5 min (Figure 3B).
Having observed that the physical properties of synthetic material 1 matched those of the endogenous and reference material, we next sought to determine whether synthetic material 1 also carried the biological properties of RvD5 n-3 DPA .We recently found that RvD5 n-3 DPA activates the orphan receptor GPR101 to exert its biological activities. 15Therefore, we assessed whether synthetic 1 displayed agonistic activities with respect to this receptor.Using an impedance-based assay, we observed that synthetic 1 potently activated GPR101 in the 0.1 to 10 nM range, as denoted by a change in impedance in cells overexpressing the receptor (Figure 4).
We next evaluated whether synthetic 1 regulated human phagocyte responses by assessing the ability of this molecule to upregulate the clearance of bacterial and fungal particles by human neutrophils and macrophages.Here we observed that SPM 1 upregulates phagocytosis of fluorescently labeled E. coli, Staphylococcus aureus, and yeast cell wall-derived zymosan A particles in both cell types, displaying a characteristic bell-shaped dose−response (Figure 5A,B).Furthermore, we observed that synthetic 1 also regulates the expression of macrophage phenotypic markers (Figure 5C), as human macrophages differentiated in the presence of synthetic 1 showed lower expression of markers linked with an inflammatory phenotype, such as CD142 and CD80, while it upregulated expression of proteins linked with tissue protection, such as Arginase-1, MerTK, and CD163 (Figure 5D).Altogether, the results presented in Figures 4 and 5 reveal that RvD5 n-3 DPA (1) possesses potent pro-resolution and anti-inflammatory actions as well as agonism toward GPR101.The regulation of neutrophils and macrophages to phagocytose bacteria that SPM 1 shows is of great interest in drug discovery efforts based on resolution pharmacology.
To obtain synthetic RvD5 n-3 DPA (1) for LC/MS-MS and receptor studies, synthetic 2 (10 μg) was incubated with 1 N LiOH (50 μL) in THF (0.5 mL) for 2 h at rt, and then H 2 O (15 μL) was added and THF evaporated using a gentle stream of nitrogen.The obtained material 1 was used as such.Reference material was obtained as detailed in ref 15.The identity of RvD5 n-3 DPA in the synthetic and biological matrices was determined using MRM with signature parent ion (Q1, m/z 361) and characteristic daughter ions (Q3, m/z 199, 143 or 263) coupled with an enhanced product ion (EPI) scan.For synthetic 1, UV data were in accord with the literature.7b,15 Biological Experiments.The experiments strictly adhered to UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986) and Laboratory Animal Science Association (LASA) Guidelines (Guiding Principles on Good Practice for Animal Welfare and Ethical Review Bodies, 3rd Edition, 2015) and according to protocols detailed in a UK Home Office approved protocol (P998AB295).
Impedance Assays.GPR101 receptor activation by synthetic RvD5 n-3 DPA (1) was assessed by monitoring impedance changes across CHO cell monolayers using an xCelligence RTCA DP system (ACEA Biosciences).GPR101-overexpressing CHO cells were plated 1 day prior to experiments at 80,000 cells per well in impedance measurement plates (E-Plate16; ACEA Biosciences).Cells were then washed with serum-free DMEM/F-12 medium, and 0.1, 1, or 10 nM of synthetic 1 or vehicle control (0.05% ethanol) in DMEM/F-12 medium was added to cells.Real-time changes in impedance were assessed over a 30 min interval.
PBMC and Neutrophil Isolation from Whole Blood.Venous peripheral blood was collected from healthy volunteers after giving written consent in accordance with a Queen Mary Ethics of Research Committee-approved (QMERC22.331)study proposal.Human peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated by Histopaque-1077 density centrifugation as follows: red blood cells (RBCs) were sedimented using 6% (w/v) dextran (MW 425,000−575,000; Sigma) followed by incubation at room temperature for 20 min.RBC-depleted upper layers were transferred to 50 mL tubes containing Histopaque-1077 (Sigma) and centrifuged for 30 min at 400g without a brake to separate PBMCs and neutrophil layers.Remaining RBCs in the neutrophil layer were lysed by incubating in 9 volumes of ice-cold ddH 2 O for 30 s followed by addition of 1 volume 10× Hank's balanced salt solution (HBSS).Cells were enumerated using a hemacytometer with Turk's stain and directly used in experiments (for neutrophils), or PBMCs were seeded in 10 cm plates at 30 million cells per plate and differentiated to macrophages by incubation in RPMI-1640 medium containing 10% human serum (Sigma) and 20 ng/mL GM-CSF (Peprotech) for 7 days.

Figure 2 .
Figure 2. MS/MS fragmentation spectrum of the synthetic material of 1 matched that of the reference spectrum for biologically produced RvD5 n-3 DPA (1).The tandem mass spectrum of synthetic 1 was compared with that of reference material using a Sciex QTRap 6500+ and Sciex OS Library Match function.

Figure 3 .
Figure 3. Chromatographic properties of synthetic 1 matched those of biogenetic RvD5 n-3 DPA .Multiple reaction chromatograms for selected ion pairs m/z 361 > 199 for (A) human serum and (B) mouse exudates.Top panels report the traces for the biological sample, while center panels report the chromatographic trace for the synthetic 1, and bottom panels report the co-injection of the synthetic with the biological materials.Peaks highlighted in blue shading correspond to the RvD5 n-3 DPA .Results are representative of n = 3 determinations for human serum samples and n = 3 mice for exudate samples.

Figure 4 .
Figure 4. Synthetic 1 activates human GPR101.GPR101-expressing CHO cells were incubated with the indicated concentrations with synthetic 1 or vehicle (DMEM/F-12 medium with 0.05% EtOH), and impedance was measured for 30 min using the xCelligence RTCA DP system.Results are representative of three independent experiments, with four replicates per experiment.
AuthorsKarina Ervik − Jesmond Dalli − Lipid Mediator Unit, Center for Biochemical Pharmacology, William Harvey Research, Institute, Barts and Journal of Natural Products